Paramyxoviruses have a natural tropism for the respiratory tract and induce a high level of local and systemic immunity. In other projects, we are actively developing live attenuated vaccines for a number of paramyxoviruses. We also have been interested in evaluating some of these attenuated derivatives for a second use, namely as vectors for expressing the protective antigens of highly pathogenic agents as potential vaccines. To date, we have focused on the human parainfluenza viruses (HPIVs). These are pediatric (for the most part) pathogens that replicate in the superficial cells of the respiratory tract and do not spread significantly beyond that site, which should be a positive factor with regard to vector safety. In addition, since many pathogens use the respiratory tract as a portal for entry and egress, it is important to develop vectors suited for direct intranasal immunization Previously, we evaluated this strategy using an existing live-attenuated vaccine virus, BHPIV3, which is being developed as an intranasal pediatric vaccine against HPIV3. We used BHPIV3 as a vector to express the structural proteins of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) individually and in combinations. Using the hamster model, we showed that the spike S glycoprotein is the only significant neutralization and protective antigen among the SARS structural proteins. We also showed that the BHPIV3-S recombinant was immunogenic and protective as an intranasal vaccine against SARS-CoV in African green monkeys, an animal model that is anatomically and phylogenetically more closely related to humans. This validated the strategy of intranasal immunization against a highly pathogenic agent. More recently, we evaluated whether intranasal inoculation with an HPIV3-vectored vaccine can induce protective immunity against Ebola virus (EV), which is an agent of severe viral hemorrhagic fever and causes a fulminating viremia. The virus is highly contagious and very lethal and can be transmitted by contact and by the aerosol route. These features make EV a potential weapon for bio-terrorism and biological warfare. Wild type recombinant HPIV3 was modified to express the EV structural glycoprotein (GP) alone (HPIV3/EboGP) or together with the EV nucleoprotein (NP) (HPIV3/EboGP-NP). Expression of EV GP by these recombinant viruses resulted in its incorporation into virus particles at a level that was approximately 15% that of the vector glycoproteins. Unexpectedly, EV GP appeared to be functional for mediating infection of the vector particle. This was indicated by the finding that HPIV3/EboGP had acquired resistance to neutralization in vitro by HPIV3-specific antibodies, and had gained sensitivity to neutralization by EV-specific antibodies. Indeed, it appeared to be more sensitive to EV-specific antibodies that to those specific for HPIV3. Guinea pigs infected with a single intranasal inoculation of 100,000 PFU of HPIV3/EboGP or HPIV3/EboGP-NP showed no apparent signs of disease. This animal model is highly sensitive to EV infection, such that a single PFU contains 400 lethal dose 50% units. Thus, the lack of disease associated with this high dose indicates that EV GP did not confer increased pathogenesis to the HPIV3 vector. This is an informative precedent in which the expression and incorporation of an attachment/penetration protein from a highly virulent virus did not confer increased pathogenesis to a vector. The immunized animals developed a strong humoral response specific to the two EV proteins. When these animals were challenged with a highly lethal intraperitoneal injection of 1000 PFU of EV, there were no outward signs of disease, no viremia or detectable EV antigen in the blood, and no evidence of infection in the spleen, liver, and lungs. In contrast, all of the control animals died or developed severe EV disease following challenge. The highly effective immunity achieved with a single vaccine dose suggests that intranasal immunization with live vectored vaccines based on recombinant respiratory viruses can be effective in inducing protective responses against severe systemic infections, such as those caused by hemorrhagic fever agents. We also evaluated these two HPIV3 constructs in Rhesus monkeys in parallel with a third construct, namely one engineered to express EV GP in combination with the cytokine adjuvant granulocyte macrophage colony stimulating factor (GM-CSF). These were administered by the respiratory route to Rhesus monkeys and evaluated for immunogenicity and protective efficacy against a highly lethal intraperitoneal challenge with EV. A single immunization with any construct expressing GP was moderately immunogenic against EV and protected 88% of the animals against severe EV disease, whereas two doses were highly immunogenic and the animals were free of disease signs or detectable EV challenge virus. These data suggest the feasibility of intranasal immunization against severe viral hemorrhagic fevers. The immunogenicity and protective efficacy of the vectored vaccine did not appear to be enhanced by co-expression of NP or GM-CSF. The vaccines described above are candidates for further evaluation as pediatric vaccines. However, they might not be effective in adults due to the high seroprevalence against HPIV3 in that population. We plan to evaluate this by testing these vaccines in animals that have been infected with HPIV3 one year earlier. A second strategy to overcome seroprevalence is to use a vector that does not usually infect humans and is antigenically distinct from common human viruses. NDV is an avian pathogen that fits these criteria. There is serological evidence that bird handlers can be infected, but infection apparently does not cause significant disease. NDV exists naturally in a variety of strains that exhibit a wide spectrum of virulence in birds, ranging from highly virulent (velogenic), to moderate virulence (mesogenic), to low virulence (lentigenic). Two strains were evaluated: the attenuated vaccine strain LaSota (NDV-LS) that replicates mostly in the chicken respiratory tract and the Beaudette C (NDV-BC) strain of intermediate virulence that produces mild systemic infection in chickens. A recombinant version of each virus was modified by the insertion, between the P and M genes, of a gene cassette encoding the HPIV3 hemagglutinin-neuraminidase (HN) protein, a test antigen with considerable historic data. The recombinant viruses were administered to African green monkeys (NDV-BC and NDV-LC) and rhesus monkeys (NDV-BC only) by the combined intranasal and intratracheal routes at a dose of 10(6.3) PFU per site, with a second equivalent dose administered 28 days later. Little or no virus shedding was detected in nasal/throat swabs or tracheal lavages following either immunization with either strain. In a separate experiment, direct examination of lung tissue confirmed a highly attenuated, restricted pattern of replication by parental NDV-BC. The serum antibody response to the foreign HN protein induced by the first immunization with either NDV vector was somewhat less than that observed following a wild type HPIV3 infection, whereas the titer following the second dose exceeded that observed with HPIV3 infection even though HPIV3 replicates much more efficiently than NDV in these animals. NDV appears to be a promising vector for the development of vaccines for humans to control localized outbreaks of emerging pathogens. We have constructed NDV recombinants that express the SARS-CoV S protein or EV GP for evaluation in the future. We also will evaluate additional avian paramyxoviruses as potential vectors.